Sequencing Genomes Propojený obrázek nelze zobrazit. Příslušný soubor byl pravděpodobně přesunut, přejmenován nebo odstraněn. Ověřte, zda propojení odkazuje na správný soubor a umístění. Human Genome Project: History, results and impact MUDr. Jan Pláteník, PhD. (December 2020) Beginnings of sequencing • 1965: Sequence of a yeast tRNA (80 bp) determined • 1977: Sanger’s and Maxam & Gilbert’s techniques invented • 1981: Sequence of human mitochondrial DNA (16.5 kbp) • 1983: Sequence of bacteriophage T7 (40 kbp) • 1984: Epstein & Barr‘s Virus (170 kbp) 1 Homo sapiens • 1985-1990: Discussion on human genome sequencing • “dangerous” - “meaningless” - “impossible to do” • 1988-1990: Foundation of HUMAN GENOME PROJECT • International collaboration: HUGO (Human Genome Organisation) • Aims: – genetic map of human genome – physical map: marker every 100 kbp – sequencing of model organisms (E. coli, S. cerevisiae, C. elegans, Drosophila, mouse) – find all human genes (estim. 60-80 tisíc) – sequence all human genome (estim. 4000 Mbp) by 2005 Other genomes • July 1995 : Haemophilus influenzae (1.8 Mbp) ... First genome of independent organism • October 1996: Saccharomyces cerevisiae (12 Mbp) ... First Eukaryota • December 1998: Caenorhabditis elegans (100 Mbp) ... First Metazoa 2 May 1998: • Craig Venter launches private biotechnology company CELERA GENOMICS, Inc. and announces intention to sequence whole human genome in just 3 years and 300 mil. USD using the whole- genome shotgun approach. • The publicly funded HGP in that time: sequenced cca 4 % of the genome March 2000: • Celera Genomics & academic collaborators publish draft genome of Drosophila melanogaster (cca 2/3 from 180 Mbp) • ... whole-genome shotgun is feasible for large genomes as well • ... ... Human genome: competition between Human Genome Project and Celera Genomics 3 International Human Genome Sequencing Consortium (Human Genome Project, HGP) • Open to co-operation from any country • 20 laboratories from USA, Great Britain, Japan, France, Germany and China • About 2800 workers, main coordinator: Francis Collins, NIH • Publicly funded (about 3 billion USD) • Approach: clone-by-clone • Results: duty to upload on internet within 24 hours (the Bermuda rule). Clone-by-clone genomic DNA fragments cca 150,000 bp cloning in BAC ( bacterial artificial chromosome ) clones positioned in the genome using physical maps (STS - sequence tagged site , fingerprint - cleavage by restrictases) digestion of every clone to short fragments cca 500 bp sequencing assembly of each clone sequence with computer 4 Celera Genomics, Inc. • Private biotechnology company, based in Rockville, Maryland, USA. President Craig Venter. • Investments into automation and computer processing, few dozens of employees • Approach: whole-genome shotgun + utilised publicly shared data from HGP. • Results: raw data temporarily available at company www site, but all other updates and annotations for commercial purpose. Whole-genome shotgun genomic DNA fragments 2, 10, 50 kbp cloned in plasmids E.coli sequencing sequence assembly using sophisticated computer algorithms 5 February 2001: • International Human Genome Sequencing Consortium publishes draft of human genome in Nature (Feb. 15 th 2001) • Draft: 90 % euchromatin (2.95 Gbp, whole genome 3.2 Gbp). 25 % definitive. • Celera Genomics, Inc. publishes human genome sequence in Science (Feb. 16 th 2001) • Sequence of euchromatin (2.91 Gbp) Advance in sequencing 1985: 500 bp /lab and day – still the Sanger dideoxynucleotide technique, but – capillary electrophoresis instead of gel – fluorescence markers instead radioactivity – full automatisation & robotisation – computer power 2000: 175,000 bp /day (Celera) 1000 bp/sec. (HGP) 6 Sequencing continues... • Human genome now: Definitive version announced 14/4/2003 …50 years since DNA double helix. The reference sequence still being updated. • Fugu rubripes: draft of genome in August 2002 • Mouse: • Celera Genomics: draft in June 2001 • Mouse Genome Sequencing Consortium: Nature, December 2002 • Laboratory rat: draft in March 2004 • Chimpanzee: September 2005 • … and many other genomes: malaria (the cause Plasmodium falciparum and carrier Anopheles gambiae), zebrafish, rice, dog, cattle, sheep, pig, chicken, honeybee, mammoth etc. Research in “postgenomic” age • New approaches to study genes & proteins: • GENOMICS ... analysis of whole genome and its expression • PROTEOMICS ... analysis of whole proteome, i.e. all proteins in given tissue or organism • BIOINFORMATICS ... processing, analysis and interpretation of large data sets (NA or protein sequences, gene arrays, 3D protein structures etc. Experiments in silico • Rapid development of new technologies: • e.g. DNA Microarray - expression of thousands of genes can be studied simultaneously 7 DNA Microarray (“ DNA chip”) Single Nucleotide Polymorphism (SNP) A G A G T T C T G C T C G A G G G T T C T G C G CG Occurs on average in one base per 1000 bp, i.e. in 0.1 % of human genome About 10 millions of SNPs with occurrence > 1% Coding/non-coding Protein structure changed/unchanged 8 International HapMap Project • Further international collaboration 2002-2009 • Genotyping and sequencing of DNA from 270 people from four different populations (USA, Nigeria, Japan, China) • Aims at finding • all important human SNPs (about 10,000,000) • their stable combinations (haplotypes) • Tag SNP for each haplotype • Data publicly available for further exploration Human genetic variation • Two unrelated humans have 99.5% of genome identical • Single Nucleotide Polymorphisms: 0.1% • Copy number variation (insertions, deletions, duplications): 0.4% • Variable number tandem repeats (…DNA fingerprinting in forensics) • Epigenetics (methylation) 9 Second-generation sequencers: E.g. Illumina Co., XII/2008: • One run (3 days) of Genome Analyzer made by Illumina Inc. = 60 years of work of ABI 3730xl (used by Celera Genomics) • Cost of one human genome sequencing: 40-50,000 $ • First individual human genomes sequenced: • 2007: Craig Venter, James Watson – both genomes published in the internet … and third-generation sequencers Graph: Nature 458, 719-724 (2009). Obtained from http://genome.wellcome.ac.uk 10 Next-Generation Sequencing (NGS) • 454 pyrosequencing • Sequencing by synthesis (Illumina) • SOLiD sequencing by ligation • Ion Torrent semiconductor sequencing • DNA nanoball sequencing • Heliscope single molecule sequencing • Single molecule real time (SMRT) sequencing • Nanopore DNA sequencing Archon X Prize for Genomics $ 10,000,000 Announced in 2006. For the first team that succeeds in sequencing of 100 individual human genomes within 30 days in certain requested quality and cost below $1,000 per one genome. 11 Archon X Prize for Genomics $ 10,000,000 Announced in 2006. For the first team that succeeds in sequencing of 100 individual human genomes within 30 days in certain requested quality and cost below $1,000 per one genome. (Cost to sequence a human genome according to NHGRI, Wikimedia Commons) 12 Next-Generation Sequencing (NGS) Current technology, e.g Illumina HiSeq 3000 or HiSeq 4000: Sequencing by synthesis (SBS) Up to 400 Gb/day …12 human genomes or 96 exomes in 3.5 days www.illumina.com https://www.youtube.com/watch?v=womKfikWlxM • Sequencing all cca 1.5 million of known eukaryotic species on Earth (… all plants, animals, protozoa and fungi, so far <0.2 % sequenced) • Launched 1/11/2018, expected to take 10 years, 4.7 billion USD. 13 Human Genome Project: Results The Human Genome Fig. from Bolzer et al. 2005, PLoS Biol. 3(5): e157 DOI: 10.1371/journal.pbio.0030157 Haploid genome: 3 billion base pairs divided to 23 chromosomes • 1 meter of DNA if extended • 750 Mb (1 CD) • 2 million standard printed pages (50 letters/line, 30 lines/page) 14 DNA in cell nucleus Nucleus of typical human cell has diameter 5-8 μm and contains 2 m of DNA Comparable to a tennis ball into which 20 km of thin thread has been neatly packed. Classification of eukaryotic genomic DNA: • Degree of condensation: • Euchromatin • Heterochromatin (cca 10%, sequencing difficult) • Repetitivity: • Highly repetitive • Moderately repetitive • Non-repetitive (single-copy) • Function: • Structural (centromeres, telomeres) • Coding protein • Transcribed to noncoding RNA (introns, rRNA, tRNA, miRNA etc.) • Transposons • Regulatory sequences • Junk…? 15 Experiments with denaturation & reassociation of DNA: Rapid reassociation (10-15%): - highly repetitive DNA Intermediate reassociation (25- 40%): - moderately r epetitive DNA Slow reassociation (50- 60%): - non-repetitive (single copy) DNA Fig: Lodish, H. et al.: Molecular Cell Biology (3rd ed.), W.H.Freeman, New York 1995. Classification of eukaryotic genomic DNA: • Highly repetitive (simple-sequence DNA): • All heterochromatin (centromeres, telomeres, 8% of genome, yet unsequenced) • Minisatellites (3% of euchromatin) • Moderately repetitive: • Tandemly repeated genes for rRNA, tRNA and histones (more identical copies to achieve high transcription efficiency, e.g. rRNA genes in eukaryotes >100 copies) • Transpozons • Non-repetitive: • Protein genes • Genes for noncoding RNA • Regulatory sequences 16 Eukaryotic GENE Fig: Murray, R.K. et al.: Harperova biochemie, Appleton & Lange 1993, in Czech H&H 2002. Genes are not placed evenly in genome • Big differences among chromosomes: • chromosome 1: 2,057 coding genes • chromosome Y: 65 coding genes • Regions rich in genes (“cities”) - more C and G • Regions poor in genes (“deserts”) - more A and T, up to 3 Mb! • CpG islands -